\appendix

\chapter{Recipies}

\section{Buffers}

\subsection{\acf{DMEM}/10\% \acf{CS}}
\label{recp:DMEM10CS}

\begin{table} [h]
\caption[\acf{DMEM}/10\% \acf{CS}]{The recipie for acf{DMEM}/10\% \acf{CS}}
\begin{center}

\begin{tabular}{| l |c |c |r |}
\hline
  Reagent & Amount & Vendor and Order No. \\
\hline
	\ac{DMEM} & 500 ml & Gibco, 41965-039) \\
    \ac{PS} & 5 ml & (Sigma, P0781) \\
    \ac{CS} & 56 ml & (Gibco cat 16010-084)	 \\
\hline
\end{tabular}
\end{center}
\end{table}




\ac{PS}: All media is supplemented with streptomycin sulfate (f.c. 0.1 mg/ml) and penicillin G (f.c.100 U/ml).  Use 100 x solution (10,000 units penicillin and 10 mg streptomycin per ml): Sigma P0781  



%\section{Two-dimensional gel electrophoresis - mini-gels}
%
%\subsubsection{Rehydration Solution}
%
%\label{app:rehydration_soln}
%\begin{table} [ht]
% \caption[Rehydration stock solution]{Rehydration stock solution. The \ac{IPG} strips are required to be rehydrated prior to sample application. DTT and IPG Buffer or Pharmalyte are added just prior to use. 7 mg of DTT are added per 2.5 ml aliquot of rehydration stock solution and stored at -20$^o$C.}
% \label{t:rehydration_soln}
%\begin{center}
%\begin{tabular}{|c|c|c|} 
%\hline
%  Reagent 		 & Final Concentration 	& Amount  \\ 
%\hline
%  Urea (FW 60.06) 	& 8 M 			& 12 g\\ \vline
%  CHAPS 		& 2\% (w/v) 		& 0.5 g \\
%Bromophenol blue 	& 0.002\% (w/v) 	& 50 $\mu$l \\
%Double distilled H$_{2}$O & - 			& to 25 ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%\subsection{Protein load}
%\begin{table} [h]
% \caption[Protein loads in micro-grams on IPG strips for silver and Coomassie staining]{Protein loads in micro-grams ($\mu$g) on IPG strips for silver and Coomassie staining. Each detection, or staining method requires different protein loads due to differeing levels of sensitivity.}
% \label{t:proteinLoad}
%\begin{center}
%
%
%
%\begin{tabular}{| l |c| c| r |}
%\hline
%  Length (cm) & pH Interval & Silver & Coomassie \\
%\hline
%  7 & 3-10 & 3 & - \\
%  7 & 6-11 & 8 & - \\
%7 & 4-7 & 4 & - \\
%7 & 6.2-7.5 & 8 & - \\
%7 & 3-5.6 NL & 8 & - \\
%24 & 4-7 & - & 500 \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%\section{Two-dimensional gel electrophoresis - large-gels}
%\subsection{Equilibration with reduction and alkylation of sample}
%
%\begin{table} [h]
% \label{t:sds_equil_buffer}
%\caption[SDS Equilibration Buffer]{SDS Equilibration Buffer. The buffer used to equilibrate the \ac{IPG} strip in the buffer and pH environment prior to second dimension separation.}
%\begin{center}
%
%\begin{tabular}{| l |c |c |r |}
%\hline
%  Reagent & Final concentration & Amount \\
%\hline
%Tris-HCl, pH 8.8 & 50 mM & 10.0 ml \\
%    Urea (FW 60.06) & 6M & 72.07 g \\
%    Glycerol (87\% v/v) & 30\% (v/v) & 69 ml \\
%    SDS (FW 288.38) & 2\% (w/v) & 4.0 g \\
%    Bromophenol blue & 0.002\% (w/v) & 400 $\mu$l\\
%    Double distilled H$_{2}$O &  & to 200 ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%\begin{table} [ht]
%\caption[NuPAGE agarose sealing solution]{NuPAGE Agarose sealing solution. All the reagents were added to a 500 ml Erlenmeyer flask and heated in a microwave until dissolved. Aliquots of 2 ml were stored at room temperature.}
% \label{t:Nupage_ass}
%\begin{center}
%
%\begin{tabular}{| l |c |c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Final concentration}\\
%\hline
%1xNuPAGE MOPS SDS  &  100 ml \\
%Running Buffer &  \\
%SDS Running Buffer &   \\
%  Agarose (NA or M)& 0.5\%  \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%\begin{table} [ht]
% \label{t:bromoblue}
%\caption{Bromophenol blue stock solution}
%\begin{center}
%
%\begin{tabular}{| l |c| c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Final concentration} & \textbf{Amount} \\
%\hline
%  Bromophenol blue & 1\% & 100 mg \\
%  Tris-base & 50 mM & 60 mg \\
%Double distilled H$_{2}$O &  & to 10 ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\begin{table} [ht]
%\caption{40$\%$ Acrylamide Resolving gel}
% \label{t:gel}
%\begin{center}
%
%\begin{tabular}{| l| c| c| r |}
%\hline
%  \textbf{Reagent}  & \textbf{Amount} \\
%\hline
%40\% Acrylamide/Bis (w/v) & 30ml \\
%1.5M Tris-HCL & 25ml \\
%10\% SDS & 1.0ml \\
%Distilled deionised water & 43.5ml \\
%TEMED & 50$\mu$l\\
%10\% APS & 500$\mu$l\\
%\hline
%Total Volume & 100ml \\ 
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\begin{table} [ht]
%\caption[SDS Agarose sealing solution]{SDS Agarose sealing solution. All the reagents were added to a 500 ml Erlenmeyer flask and heated in a microwave until dissolved. Aliquots of 2 ml were stored at room temperature.}
% \label{t:sds_ass}
%\begin{center}
%
%\begin{tabular}{| l |c |c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Amount }\\
%\hline
%SDS Electrophoresis buffer\footnotetext{\ref{t:sds_elec_buffer}}  & 100 ml \\
%  Agarose (NA or M) & 0.5 g \\
%  Bromophenol blue  & 200 $\mu$l \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%
%\begin{table} [ht]
% \label{t:sds_elec_buffer}
%\caption{SDS Electrophoresis buffer}
%\begin{center}
%
%\begin{tabular}{| l| c| c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Final concentration} & \textbf{Amount} \\
%\hline
%Tris base (FW 121.1) & 25 mM & 30.3 g \\
%Glycine (FW 75.07) & 192 mM & 144.0 g \\
%SDS (FW 288.38) & 0.1\% (w/v) & 10.0 g \\
%Double distilled H$_{2}$O & & to 10 l \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\section{Western blot}
%
%
%\begin{table} [ht]
% \label{t:transfer_buffer}
%\caption{1xNuPAGE Transfer Buffer}
%\begin{center}
%
%\begin{tabular}{| l |c| c |r |}
%\hline
%  Reagent & Volume \\
%\hline
%NuPAGE Transfer Buffer (20X) & 50ml\\
%NuPAGE Antioxidant & 1ml \\
%Methanol & 200ml \\
%Double distilled water & to 849ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\section{Post-transfer analysis}
%\begin{table} 
% \label{t:10XTBS}
%\caption{10x\acf{TBS}}
%\begin{center}
%
%\begin{tabular}{| l |c| c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Amount} \\
%\hline
%200 mM Tris & 96.8g / 4l \\
%1.5 M NaCl & 350.8g / 4l \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\begin{table} 
% \label{t:buffer_A}
%\caption{Blocking buffer A}
%\begin{center}
%
%\begin{tabular}{| l |c| c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Amount} \\
%\hline
%Marvel Milk powder & 5g \\
%1XTBS (table \ref{t:10XTBS}  & 100ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%\begin{table} 
% \label{t:buffer_B}
%\caption{Blocking buffer B}
%\begin{center}
%
%\begin{tabular}{| l |c |c |r |}
%\hline
%  \textbf{Reagent} & \textbf{Amount} \\
%\hline
%Marvel Milk powder & 0.5g \\
%1XTBS (table \ref{t:10XTBS}  & 50ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%
%
%\begin{table} 
% \label{t:TTBS}
%\caption{\acf{TTBS}}
%\begin{center}
%
%\begin{tabular}{| l |c| c| r |}
%\hline
%  \textbf{Reagent} & \textbf{Amount} \\
%\hline
%Tween 20 & 2.4ml \\
%1xTBS (table \ref{t:10XTBS}  & 800ml \\
%\hline
%\end{tabular}
%\end{center}
%\end{table}
%% 
%% 
%% \begin{table} [h]
%%  \label{t:tris}
%% \caption{Resolving Gel Buffer (Tris-HCL), Filtered through a 0.45 $\mu$m filter. Stored at 4$^o$C.}
%% \begin{center}
%% 
%% \begin{tabular}{| l| c| c| r |}
%% \hline
%%   Reagent & Final concentration & Amount \\
%% \hline
%% Tris base (FW 121.1) & 1.5 M & 181.7 g \\
%% Double distilled H$_2$O & & 750 ml \\
%% HCl (FW 36.46) & & adjust to pH 8.8 \\
%% Double distilled H$_2$O & & to 1 l \\
%% \hline
%% \end{tabular}
%% \end{center}
%% \end{table}
%
%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%
%\chapter{Proteomics investigation}
%
%\section{Mascot search results: mini-gels}
%\label{app:mascot_sum_mini}
%
%http://homepages.cs.ncl.ac.uk/frank.gibson/ \\
%Thesis/Appendix/Proteomics/Protein$\_$Summary$\_$Report$\_$(F262$\_$z$\_$0001.pdf
%
%
%\section{Mascot search results: large-gels}
%\label{app:mascot_sum_large}
%
%http://homepages.cs.ncl.ac.uk/frank.gibson/Thesis/ \\
%Appendix/Proteomics/Protein$\_$Summary$\_$Report$\_$(FG$\_$7v8$\_$4$\_$0001.dat - SpecView).pdf
%
%
%
%
%
%
